Responsiveness of the NIL Col-S2 and <i>mpk12</i> mutants to stomatal opening and closing stimuli.

<p>(A) Stomatal opening induced by 100 ppm CO₂ in whole plants (58 min after induction; <i>n</i> = 12–13). (B) Light-induced stomatal opening inhibited by 2.5 μM ABA in whole plants (24 min after induction; <i>n</i> = 16–18). (C) Stomatal closure induced by 800 ppm CO₂ in whole plants (10 min after induction; <i>n</i> = 12–13). (D) Stomatal closure induced by spraying whole plants with 5 μM ABA solution (24 min after induction; <i>n</i> = 12–14). (E) MPK12 is required for the bicarbonate (HCO₃^-)-induced slow type anion channel activation in guard cell protoplasts. Upper panels show typical whole guard cell protoplast recordings with 11.5 mM free HCO₃^- added to the pipette solution, and lower panels show average steady-state current-voltage relationships for wild-type (Col-0), NIL Col-S2, and <i>mpk12-4</i> after treatment with mock or 11.5 mM HCO₃^- (<i>n</i> = 4–8 per line and treatment). Small letters (A, C) and asterisks (B, D) indicate statistically significant differences according to one-way ANOVA and two-way ANOVA with Tukey HSD for unequal sample size post hoc tests (<i>p</i> < 0.05), respectively. Error bars mark ± SEM. The raw data for panels A–E can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000322#pbio.2000322.s011" target="_blank">S1 Data</a>.</p

MPK12 interacts with HT1.

<p>(A) Split-ubiquitin yeast two-hybrid assay on the SD-LeuTrp plate (left and middle panels) indicates the presence of both bait and prey plasmids; X-gal overlay assay (middle) and growth assay on the SD-LeuTrpHisAde plate (right) show HT1 interaction with MPK12 that is similar to the positive control (pAI-Alg5). Only weak or no interaction was detected with MPK12 G53R and MPK11, similar to the negative control (pDL2-Alg5). (B) Quantitative β-galactosidase assay from pools of ten colonies each. Activities are shown as the percentage of the positive control (± SEM; <i>n</i> = 3). (C) High-magnification (63x objective) BiFC images from a single infiltrated <i>N</i>. <i>benthamiana</i> leaf with identical confocal microscopy acquisition settings. Scale bar = 50 μm. (D) Ratiometric BiFC shows weaker interaction of MPK12 G53R than MPK12 with HT1, while MPK11 exhibits a weak interaction with HT1. The plasma membrane–localized SLAC1-CFP was used as an internal control. Eighteen images (from three leaves) of each construct set were analyzed. (E) Western blot together with Coomassie staining of proteins extracted from BiFC samples used for confocal imaging and controls with single construct shows expression of all fusion proteins. (F) Steady-state stomatal conductance of Col-S2 <i>ht1-2</i>, <i>mpk12-4 ht1-2</i>, and Col-S2 <i>abi1-1</i> (<i>ABA insensitive 1–1</i>) double mutants (mean ± SEM, <i>n</i> = 11–13). Experiments were repeated at least three times. Letters in B, D, and F denote statistically significant differences with one-way ANOVA and Tukey HSD post hoc test for equal B, D, or unequal F sample size. The raw data for panels B, D, and F can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000322#pbio.2000322.s011" target="_blank">S1 Data</a>.</p

Regulation of HT1 by MPK12.

<p>(A) Inhibition of HT1 kinase activity in vitro by different versions of MPK12 (MPK12 G53R—Cvi-0 version of MPK12; MPK12 K70R—inactive kinase; MPK12 Y122C—hyperactive kinase). Upper panel: autoradiography of the SDS PAGE gel; lower panel: Coomassie-stained SDS PAGE. Reaction mixture was incubated for 30 min. (B) Casein phosphorylation by HT1 with different MPK12 concentrations (mean ± SEM; <i>n</i> = 3). The raw data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000322#pbio.2000322.s011" target="_blank">S1 Data</a>. (C) Kinase-dead HT1 K113M was not in vitro phosphorylated by different versions of MPK12, and only MPK12 and MPK12 (Y122C) display clear autophosphorylation activities.</p

Stomatal conductance of the NIL Col-S2, <i>mpk12</i> mutants, and complementation lines.

<p>(A) Diurnal pattern of stomatal conductance with 12 h/12 h light–dark periods (<i>n</i> = 13–16). (B) Instantaneous water use efficiency (WUE) measured as an average of daytime light period from 09:00 to 17:00 (<i>n</i> = 13–16). (C) Stomatal conductance of Cvi-0 transformed with Col-0 <i>MPK12</i> driven by its native promoter in T2 generation (<i>n</i> = 9). (D) Stomatal conductance of Col-S2 complementation line in T2 generation transformed with Col-0 <i>MPK12</i>, driven by its native promoter (<i>n</i> = 5–8). (E) Stomatal conductance of T3 transformants in the <i>mpk12-4</i> background transformed with either the Col-0 or Cvi-0 version of <i>MPK12</i>, driven by its respective native promoter (<i>n</i> = 5–6). All graphs present mean ± SEM. Small letters denote statistically significant differences according to one-way ANOVA with Tukey HSD post hoc test for either unequal (B, D, E) or equal sample size (C). The raw data for panels A–E can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000322#pbio.2000322.s011" target="_blank">S1 Data</a>.</p

A conserved glycine is important for MPK4 and MPK12 function.

<p>(A) Inhibition of HT1 kinase activity in vitro by MPK4 and MPK4 G55R. Upper panel: autoradiography of the SDS PAGE gel; lower panel: Coomassie-stained SDS PAGE. Reaction mixture was incubated for 30 min. (B) Whole protein (left) and close-up (right) view of the superposition of models for MPK12 wild-type (secondary structure and surface in white) and MPK12 G53R (secondary structure in green). There is a close structural similarity between the structures except where the arginine at position 53 protrudes from the mutant protein surface and changes the loop region for the mutant. (C) Whole protein (left) and close-up (right) view of the superposition of models for MPK4 wild-type (secondary structure and surface in white) and MPK4 G55R (secondary structure in yellow). Similar to MPK12 G53R, the arginine at position 55 in MPK4 protrudes from the mutant protein surface and changes the loop region.</p

Mapping Cvi-0 ozone sensitivity and Cvi-0 and <i>cis</i> stomatal phenotypes.

<p>(A) Tissue damage after 6 h of O₃ exposure (350 ppb). Visual damage of plant rosettes (upper images) and cell death visualized with trypan blue staining (lower images). Scale bars 1 cm. (B) Stomatal conductance of Col-0, Cvi-0, and NILs (mean ± standard error of the mean [SEM], <i>n</i> = 7–12). (C) Elevated CO₂ (800 ppm) induced stomatal closure in intact whole plants (<i>n</i> = 9–10, except <i>cas-2</i> [<i>n</i> = 3]). Experiment was repeated at least three times with similar results. (D) Stomatal half-response times to elevated CO₂ (800 ppm). Error bars indicate ± SEM (<i>n</i> = 13). Pooled data from two experimental series are shown. (E) Gene model of <i>MPK12</i> (At2g46070) and <i>BYPASS2</i> (At2g46080). The deletion mutant <i>cis</i> (renamed as <i>mpk12-4</i>) has a 4,772 bp deletion (end and start indicated). Col-S2 has a G to C missense mutation at position 157 of <i>MPK12</i>, which leads to G53R substitution in MPK12. The <i>mpk12-3</i> mutant has a Syngenta Arabidopsis Insertion Library (SAIL) transfer DNA (T-DNA) insertion in the second exon of <i>MPK12</i>. White boxes refer to exons, grey boxes to introns, and black lines to intergenic regions. Small letters (B) and asterisks (D) denote statistically significant differences according to one-way ANOVA with Tukey honest significant difference (HSD) for unequal sample size (Spjotvoll & Stoline test) or Tukey HSD post hoc test, respectively. The raw data for panels B–D can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000322#pbio.2000322.s011" target="_blank">S1 Data</a>.</p